Nucleic acid molecules encoding human secreted hemopexin-related proteins

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the secreted peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the secreted peptides, and methods of identifying modulators of the secreted peptides.

FIELD OF THE INVENTION

The present invention is in the field of secreted proteins that arerelated to the hemopexin subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins that effect protein phosphorylation and nucleicacid molecules encoding such peptide and protein molecules, all of whichare useful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION

Secreted Proteins

Many human proteins serve as pharmaceutically active compounds. Severalclasses of human proteins that serve as such active compounds includehormones, cytokines, cell growth factors, and cell differentiationfactors. Most proteins that can be used as a pharmaceutically activecompound fall within the family of secreted proteins. It is, therefore,important in developing new pharmaceutical compounds to identifysecreted proteins that can be tested for activity in a variety of animalmodels. The present invention advances the state of the art by providingmany novel human secreted proteins.

Secreted proteins are generally produced within cells at roughendoplasmic reticulum, are then exported to the golgi complex, and thenmove to secretory vesicles or granules, where they are secreted to theexterior of the cell via exocytosis.

Secreted proteins are particularly useful as diagnostic markers. Manysecreted proteins are found, and can easily be measured, in serum. Forexample, a ‘signal sequence trap’ technique can often be utilizedbecause many secreted proteins, such as certain secretory breast cancerproteins, contain a molecular signal sequence for cellular export.Additionally, antibodies against particular secreted serum proteins canserve as potential diagnostic agents, such as for diagnosing cancer.

Secreted proteins play a critical role in a wide array of importantbiological processes in humans and have numerous utilities; severalillustrative examples are discussed herein. For example, fibroblastsecreted proteins participate in extracellular matrix formation.Extracellular matrix affects growth factor action, cell adhesion, andcell growth. Structural and quantitative characteristics of fibroblastsecreted proteins are modified during the course of cellular aging andsuch aging related modifications may lead to increased inhibition ofcell adhesion, inhibited cell stimulation by growth factors, andinhibited cell proliferative ability (Eleftheriou et al., Mutat Res 1991March-November; 256(2-6):127-38).

The secreted form of amyloid beta/A4 protein precursor (APP) functionsas a growth and/or differentiation factor. The secreted form of APP canstimulate neurite extension of cultured neuroblastoma cells, presumablythrough binding to a cell surface receptor and thereby triggeringintracellular transduction mechanisms. (Roch et al., Ann N.Y. Acad Sci1993 September 24;695:149-57). Secreted APPs modulate neuronalexcitability, counteract effects of glutamate on growth cone behaviors,and increase synaptic complexity. The prominent effects of secreted APPson synaptogenesis and neuronal survival suggest that secreted APPs playa major role in the process of natural cell death and, furthermore, mayplay a role in the development of a wide variety of neurologicaldisorders, such as stroke, epilepsy, and Alzheimer's disease (Mattson etal., Perspect Dev Neurobiol 1998; 5(4):337-52).

Breast cancer cells secrete a 52K estrogen-regulated protein (seeRochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secretedprotein is therefore useful in breast cancer diagnosis.

Two secreted proteins released by platelets, platelet factor 4 (PF4) andbeta-thromboglobulin (betaTG), are accurate indicators of plateletinvolvement in hemostasis and thrombosis and assays that measure thesesecreted proteins are useful for studying the pathogenesis and course ofthromboembolic disorders (Kaplan, Adv Exp Med Biol 1978; 102:105-19).

Vascular endothelial growth factor (VEGF) is another example of anaturally secreted protein. VEGF binds to cell-surface heparan sulfates,is generated by hypoxic endothelial cells, reduces apoptosis, and bindsto high-affinity receptors that are up-regulated by hypoxia (Asahara etal., Semin Interv Cardiol 1996 September; 1 (3):225-32).

Many critical components of the immune system are secreted proteins,such as antibodies, and many important functions of the immune systemare dependent upon the action of secreted proteins. For example, Saxonet al., Biochem Soc Trans 1997 May; 25(2):383-7, discusses secreted IgEproteins.

For a further review of secreted proteins, see Nilsen-Hamilton et al.,Cell Biol Int Rep 1982 September; 6(9):815-36.

Hemopexin

The novel human protein, and encoding gene, provided by the presentinvention is related to hemopexin proteins. Hemopexins are globulins(beta-glycoproteins) that are synthesized in the liver and represent1.4% if total serum protein. Each hemopexin molecule binds a single hememolecule with high affinity and transports the heme to hepatocytes fortransfer of iron. Hemopexin levels are low in individuals withhemolysis.

Due to their importance in hematological physiology, particularly inregulating transportation of heme and iron, novel humanhemopexin-related proteins/genes, such as provided by the presentinvention, are valuable as potential targets and/or reagents for thedevelopment of therapeutics to treat hematological diseases/disorderssuch as hemolysis and anemia, as well as other diseases/disorders.Furthermore, SNPs in hemopexin-related genes may serve as valuablemarkers for the diagnosis, prognosis, prevention, and/or treatment ofsuch diseases/disorders. Using the information provided by the presentinvention, reagents such as probes/primers for detecting the SNPs or theexpression of the protein/gene provided herein may be readily developedand, if desired, incorporated into kit formats such as nucleic acidarrays, primer extension reactions coupled with mass spec detection (forSNP detection), or TAQMAN PCR assays (Applied Biosystems, Foster City,Calif.).

For a further review of hemopexin, see Law et al., Genomics 3 (1), 48-52(1988); Altruda et al., J. Mol. Evol. 27 (2), 102-108 (1988); Altruda etal., Nucleic Acids Res Jun. 11, 1985;13(11):3841-59; Cai et al., Am. J.Hum. Genet. 39: A191 only, 1986; Kamboh et al., Am. Hum. Genet. 41:645-653, 1987; Lush, “The Biochemical Genetics of Vertebrates ExceptMan.” Philadelphia: W. B. Saunders (pub.) 1966; Naylor et al, Somat.Cell Molec. Genet. 13: 355-358, 1987; Roychoudhury et al., “HumanPolymorphic Genes: World Distribution.” New York: Oxford Univ. Press(pub.) 1988; Stewart et al., Ann. Hum. Genet. 35: 19-24, 1971; andTakahashi et al., Proc. Nat. Acad. Sci. 82: 73-77, 1985.

Secreted proteins, particularly members of the hemopexin proteinsubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of secreted proteins. The present invention advances the stateof the art by providing previously unidentified human secreted proteinsthat have homology to members of the hemopexin protein subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human secreted peptides and proteins that are relatedto the hemopexin protein subfamily, as well as allelic variants andother mammalian orthologs thereof. These unique peptide sequences, andnucleic acid sequences that encode these peptides, can be used as modelsfor the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate secreted proteinactivity in cells and tissues that express the secreted protein.Experimental data as provided in FIG. 1 indicates expression in thefetal brain, brain neuroblastoma cells, liver, and fetal liver.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodesthe secreted protein of the present invention. (SEQ ID NO: 1) Inaddition, structure and functional information is provided, such as ATGstart, stop and tissue distribution, where available, that allows one toreadily determine specific uses of inventions based on this molecularsequence. Experimental data as provided in FIG. 1 indicates expressionin the fetal brain, brain neuroblastoma cells, liver, and fetal liver.

FIG. 2 provides the predicted amino acid sequence of the secretedprotein of the present invention. (SEQ ID NO:2) In addition structureand functional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding thesecreted protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 10 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a secreted protein or part of a secreted protein andare related to the hemopexin protein subfamily. Utilizing thesesequences, additional genomic sequences were assembled and transcriptand/or cDNA sequences were isolated and characterized. Based on thisanalysis, the present invention provides amino acid sequences of humansecreted peptides and proteins that are related to the hemopexin proteinsubfamily, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode these secretedpeptides and proteins, nucleic acid variation (allelic information),tissue distribution of expression, and information about the closest artknown protein/peptide/domain that has structural or sequence homology tothe secreted protein of the present invention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known secreted proteins of the hemopexinprotein subfamily and the expression pattern observed. Experimental dataas provided in FIG. 1 indicates expression in the fetal brain, brainneuroblastoma cells, liver, and fetal liver. The art has clearlyestablished the commercial importance of members of this family ofproteins and proteins that have expression patterns similar to that ofthe present gene. Some of the more specific features of the peptides ofthe present invention, and the uses thereof, are described herein,particularly in the Background of the Invention and in the annotationprovided in the Figures, and/or are known within the art for each of theknown hemopexin family or subfamily of secreted proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thesecreted protein family of proteins and are related to the hemopexinprotein subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIG. 1 and genomic sequencesare provided in FIG. 3). The peptide sequences provided in FIG. 2, aswell as the obvious variants described herein, particularly allelicvariants as identified herein and using the information in FIG. 3, willbe referred herein as the secreted peptides of the present invention,secreted peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the secreted peptides disclosed in the FIG. 2, (encoded bythe nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thesecreted peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

The isolated secreted peptide can be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Experimental data as provided in FIG. 1 indicates expression in thefetal brain, brain neuroblastoma cells, liver, and fetal liver. Forexample, a nucleic acid molecule encoding the secreted peptide is clonedinto an expression vector, the expression vector introduced into a hostcell and the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the secreted peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The secreted peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a secreted peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the secreted peptide. “Operatively linked”indicates that the secreted peptide and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-terminusor C-terminus of the secreted peptide.

In some uses, the fusion protein does not affect the activity of thesecreted peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant secreted peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A secreted peptide-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the secreted peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the secreted peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A.M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D.W., ed., Academic Press. New York, 1993;Computer Analysis of Sequence Data. Part 1, Griffin, A.M., and Griffin,H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5,or 6. In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res.12(1):387(1984)) using a NWSgapdna. CMP matrix and a gap weight of 40,50, 60, 70, or 80 and a length weight of 1, 2, 3,4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of B. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thesecreted peptides of the present invention as well as being encoded bythe same genetic locus as the secreted peptide provided herein. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 11 by ePCR.

Allelic variants of a secreted peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the secreted peptide as wellas being encoded by the same genetic locus as the secreted peptideprovided herein. Genetic locus can readily be determined based on thegenomic information provided in FIG. 3, such as the genomic sequencemapped to the reference human. As indicated by the data presented inFIG. 3, the map position was determined to be on chromosome 11 by ePCR.As used herein, two proteins (or a region of the proteins) havesignificant homology when the amino acid sequences are typically atleast about 70-80%, 80-90%, and more typically at least about 90-95% ormore homologous. A significantly homologous amino acid sequence,according to the present invention, will be encoded by a nucleic acidsequence that will hybridize to a secreted peptide encoding nucleic acidmolecule under stringent conditions as more fully described below.

FIG. 3 provides information on SNPs that have been found in a geneencoding the secreted proteins of the present invention. SNPs wereidentified at 10 different nucleotide positions.

Paralogs of a secreted peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the secreted peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a secreted peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a secreted peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the secreted peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a secreted peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the secreted peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the secreted peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a secreted peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant secreted peptides can be fully functional or can lack functionin one or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as secreted protein activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of the secretedpeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a secreted peptide. Such fragmentscan be chosen based on the ability to retain one or more of thebiological activities of the secreted peptide or could be chosen for theability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe secreted peptide, e.g., active site or a substrate-binding domain.Further, possible fragments include, but are not limited to, domain ormotif containing fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in secreted peptides aredescribed in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N. Y Acad. Sci. 663:48-62(1992)).

Accordingly, the secreted peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature secreted peptide is fused withanother compound, such as a compound to increase the half-life of thesecreted peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature secreted peptide, such asa leader or secretory sequence or a sequence for purification of themature secreted peptide or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a secretedprotein-effector protein interaction or secreted protein-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, secreted proteins isolated from humans andtheir human/mammalian orthologs serve as targets for identifying agentsfor use in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the secreted protein. Experimental data asprovided in FIG. 1 indicates that secreted proteins of the presentinvention are expressed in fetal brain, brain neuroblastoma cells, andliver (as indicated by virtual northern blot analysis), as well as infetal liver (as indicated by the tissue source of the cDNA clone of thepresent invention). A large percentage of pharmaceutical agents arebeing developed that modulate the activity of secreted proteins,particularly members of the hemopexin subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in the fetal brain, brain neuroblastomacells, liver, and fetal liver. Such uses can readily be determined usingthe information provided herein, that which is known in the art, androutine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to secreted proteins that are related tomembers of the hemopexin subfamily. Such assays involve any of the knownsecreted protein functions or activities or properties useful fordiagnosis and treatment of secreted protein-related conditions that arespecific for the subfamily of secreted proteins that the one of thepresent invention belongs to, particularly in cells and tissues thatexpress the secreted protein. Experimental data as provided in FIG. 1indicates that secreted proteins of the present invention are expressedin fetal brain, brain neuroblastoma cells, and liver (as indicated byvirtual northern blot analysis), as well as in fetal liver (as indicatedby the tissue source of the cDNA clone of the present invention).

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the secreted protein, as abiopsy or expanded in cell culture. Experimental data as provided inFIG. 1 indicates expression in the fetal brain, brain neuroblastomacells, liver, and fetal liver. In an alternate embodiment, cell-basedassays involve recombinant host cells expressing the secreted protein.

The polypeptides can be used to identify compounds that modulatesecreted protein activity of the protein in its natural state or analtered form that causes a specific disease or pathology associated withthe secreted protein. Both the secreted proteins of the presentinvention and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the secreted protein. These compounds can be further screenedagainst a functional secreted protein to determine the effect of thecompound on the secreted protein activity. Further, these compounds canbe tested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the secreted protein to a desireddegree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thesecreted protein and a molecule that normally interacts with thesecreted protein, e.g. a substrate or a component of the signal pathwaythat the secreted protein normally interacts (for example, anothersecreted protein). Such assays typically include the steps of combiningthe secreted protein with a candidate compound under conditions thatallow the secreted protein, or fragment, to interact with the targetmolecule, and to detect the formation of a complex between the proteinand the target or to detect the biochemical consequence of theinteraction with the secreted protein and the target.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantsecreted proteins or appropriate fragments containing mutations thataffect secreted protein function and thus compete for substrate.Accordingly, a fragment that competes for substrate, for example with ahigher affinity, or a fragment that binds substrate but does not allowrelease, is encompassed by the invention.

Any of the biological or biochemical functions mediated by the secretedprotein can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the secreted protein can be assayed.Experimental data as provided in FIG. 1 indicates that secreted proteinsof the present invention are expressed in fetal brain, brainneuroblastoma cells, and liver (as indicated by virtual northern blotanalysis), as well as in fetal liver (as indicated by the tissue sourceof the cDNA clone of the present invention).

Binding and/or activating compounds can also be screened by usingchimeric secreted proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native secreted protein. Accordingly, a different setof signal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the secreted protein is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the secreted protein (e.g. binding partners and/or ligands). Thus,a compound is exposed to a secreted protein polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble secreted protein polypeptide is also added to themixture. If the test compound interacts with the soluble secretedprotein polypeptide, it decreases the amount of complex formed oractivity from the secreted protein target. This type of assay isparticularly useful in cases in which compounds are sought that interactwith specific regions of the secreted protein. Thus, the solublepolypeptide that competes with the target secreted protein region isdesigned to contain peptide sequences corresponding to the region ofinterest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the secreted protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofsecreted protein-binding protein found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques. For example,either the polypeptide or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin using techniques wellknown in the art. Alternatively, antibodies reactive with the proteinbut which do not interfere with binding of the protein to its targetmolecule can be derivatized to the wells of the plate, and the proteintrapped in the wells by antibody conjugation. Preparations of a secretedprotein-binding protein and a candidate compound are incubated in thesecreted protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thesecreted protein target molecule, or which are reactive with secretedprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

Agents that modulate one of the secreted proteins of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

Modulators of secreted protein activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by the secreted protein pathway, by treating cells or tissuesthat express the secreted protein. Experimental data as provided in FIG.1 indicates expression in the fetal brain, brain neuroblastoma cells,liver, and fetal liver. These methods of treatment include the steps ofadministering a modulator of secreted protein activity in apharmaceutical composition to a subject in need of such treatment, themodulator being identified as described herein.

In yet another aspect of the invention, the secreted proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the secreted protein and are involved insecreted protein activity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a secreted proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a secretedprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the secreted protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a secreted protein-modulating agent, anantisense secreted protein nucleic acid molecule, a secretedprotein-specific antibody, or a secreted protein-binding partner) can beused in an animal or other model to determine the efficacy, toxicity, orside effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal or other modelto determine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

The secreted proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in the fetal brain, brain neuroblastoma cells,liver, and fetal liver. The method involves contacting a biologicalsample with a compound capable of interacting with the secreted proteinsuch that the interaction can be detected. Such an assay can be providedin a single detection format or a multi-detection format such as anantibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered secreted protein activity incell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997 outcomes of these variations result in severetoxicity of therapeutic drugs in certain individuals or therapeuticfailure of drugs in certain individuals as a result of individualvariation in metabolism. Thus, the genotype of the individual candetermine the way a therapeutic compound acts on the body or the way thebody metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the secreted protein in which one ormore of the secreted protein functions in one population is differentfrom those in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other substrate-binding regionsthat are more or less active in substrate binding, and secreted proteinactivation. Accordingly, substrate dosage would necessarily be modifiedto maximize the therapeutic effect within a given population containinga polymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in thefetal brain, brain neuroblastoma cells, liver, and fetal liver.Accordingly, methods for treatment include the use of the secretedprotein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe secreted proteins. Antibodies can be prepared from any region of thepeptide as described herein. However, preferred regions will includethose involved in function/activity and/or secreted protein/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatsecreted proteins of the present invention are expressed in fetal brain,brain neuroblastoma cells, and liver (as indicated by virtual northernblot analysis), as well as in fetal liver (as indicated by the tissuesource of the cDNA clone of the present invention). Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in the fetal brain, brain neuroblastoma cells, liver, andfetal liver. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in thefetal brain, brain neuroblastoma cells, liver, and fetal liver. Thediagnostic uses can be applied, not only in genetic testing, but also inmonitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in the fetal brain, brainneuroblastoma cells, liver, and fetal liver. Thus, where a specificprotein has been correlated with expression in a specific tissue,antibodies that are specific for this protein can be used to identify atissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the secreted peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a secreted peptide or protein of the present invention(cDNA, transcript and genomic sequence). Such nucleic acid moleculeswill consist of, consist essentially of, or comprise a nucleotidesequence that encodes one of the secreted peptides of the presentinvention, an allelic variant thereof, or an ortholog or paralogthereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated.

Further examples of isolated DNA molecules include recombinant DNAmolecules maintained in heterologous host cells or purified (partiallyor substantially) DNA molecules in solution. Isolated RNA moleculesinclude in vivo or in vitro RNA transcripts of the isolated DNAmolecules of the present invention. Isolated nucleic acid moleculesaccording to the present invention further include such moleculesproduced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the secreted peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, MRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as MRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the secreted proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 11 by ePCR.

FIG. 3 provides information on SNPs that have been found in a geneencoding the secreted proteins of the present invention. SNPs wereidentified at 10 different nucleotide positions.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at 10 different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 11 by ePCR.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the MRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatsecreted proteins of the present invention are expressed in fetal brain,brain neuroblastoma cells, and liver (as indicated by virtual northernblot analysis), as well as in fetal liver (as indicated by the tissuesource of the cDNA clone of the present invention). Accordingly, theprobes can be used to detect the presence of, or to determine levels of,a specific nucleic acid molecule in cells, tissues, and in organisms.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes corresponding to the peptides described herein canbe used to assess expression and/or gene copy number in a given cell,tissue, or organism. These uses are relevant for diagnosis of disordersinvolving an increase or decrease in secreted protein expressionrelative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a secreted protein, such as by measuring alevel of a secreted protein-encoding nucleic acid in a sample of cellsfrom a subject e.g., MRNA or genomic DNA, or determining if a secretedprotein gene has been mutated. Experimental data as provided in FIG. 1indicates that secreted proteins of the present invention are expressedin fetal brain, brain neuroblastoma cells, and liver (as indicated byvirtual northern blot analysis), as well as in fetal liver (as indicatedby the tissue source of the cDNA clone of the present invention).

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate secreted protein nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe secreted protein gene, particularly biological and pathologicalprocesses that are mediated by the secreted protein in cells and tissuesthat express it. Experimental data as provided in FIG. 1 indicatesexpression in the fetal brain, brain neuroblastoma cells, liver, andfetal liver. The method typically includes assaying the ability of thecompound to modulate the expression of the secreted protein nucleic acidand thus identifying a compound that can be used to treat a disordercharacterized by undesired secreted protein nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the secreted protein nucleicacid or recombinant cells genetically engineered to express specificnucleic acid sequences.

Thus, modulators of secreted protein gene expression can be identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of mRNA determined. The level of expression of secretedprotein mRNA in the presence of the candidate compound is compared tothe level of expression of secreted protein mRNA in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of nucleic acid expression based on this comparison and beused, for example to treat a disorder characterized by aberrant nucleicacid expression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate secreted protein nucleic acid expression incells and tissues that express the secreted protein. Experimental dataas provided in FIG. 1 indicates that secreted proteins of the presentinvention are expressed in fetal brain, brain neuroblastoma cells, andliver (as indicated by virtual northern blot analysis), as well as infetal liver (as indicated by the tissue source of the cDNA clone of thepresent invention). Modulation includes both up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for secreted protein nucleic acid expressioncan be a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thesecreted protein nucleic acid expression in the cells and tissues thatexpress the protein. Experimental data as provided in FIG. 1 indicatesexpression in the fetal brain, brain neuroblastoma cells, liver, andfetal liver.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe secreted protein gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in secreted protein nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in secreted protein genesand gene expression products such as mRNA. The nucleic acid moleculescan be used as hybridization probes to detect naturally occurringgenetic mutations in the secreted protein gene and thereby to determinewhether a subject with the mutation is at risk for a disorder caused bythe mutation. Mutations include deletion, addition, or substitution ofone or more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the secreted protein geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a secretedprotein.

Individuals carrying mutations in the secreted protein gene can bedetected at the nucleic acid level by a variety of techniques. FIG. 3provides information on SNPs that have been found in a gene encoding thesecreted proteins of the present invention. SNPs were identified at 10different nucleotide positions. As indicated by the data presented inFIG. 3, the map position was determined to be on chromosome 11 by ePCR.Genomic DNA can be analyzed directly or can be amplified by using PCRprior to analysis. RNA or cDNA can be used in the same way. In someuses, detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a secreted protein gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantsecreted protein gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the secreted protein genein an individual in order to select an appropriate compound or dosageregimen for treatment. FIG. 3 provides information on SNPs that havebeen found in a gene encoding the secreted proteins of the presentinvention. SNPs were identified at 10 different nucleotide positions.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol secreted protein gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of secreted protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into secreted protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of secreted protein nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired secreted protein nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the secreted protein,such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in secreted protein geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredsecreted protein to treat the individual.

The invention also encompasses kits for detecting the presence of asecreted protein nucleic acid in a biological sample. Experimental dataas provided in FIG. 1 indicates that secreted proteins of the presentinvention are expressed in fetal brain, brain neuroblastoma cells, andliver (as indicated by virtual northern blot analysis), as well as infetal liver (as indicated by the tissue source of the cDNA clone of thepresent invention). For example, the kit can comprise reagents such as alabeled or labelable nucleic acid or agent capable of detecting secretedprotein nucleic acid in a biological sample; means for determining theamount of secreted protein nucleic acid in the sample; and means forcomparing the amount of secreted protein nucleic acid in the sample witha standard.

The compound or agent can be packaged in a suitable container. The kitcan further comprise instructions for using the kit to detect secretedprotein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/l 1995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the secreted proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of thesecreted protein gene of the present invention. FIG. 3 providesinformation on SNPs that have been found in a gene encoding the secretedproteins of the present invention. SNPs were identified at 10 differentnucleotide positions.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified secreted protein gene of the present inventioncan be routinely identified using the sequence information disclosedherein can be readily incorporated into one of the established kitformats which are well known in the art, particularly expression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al, Molecular Cloning. A Laboratory Manual. 2nd.ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology. Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory ,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such as kinases,appropriate secretion signals are incorporated into the vector. Thesignal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with kinases, the protein can be isolated from the host cell bystandard disruption procedures, including freeze thaw, sonication,mechanical disruption, use of lysing agents and the like. The peptidecan then be recovered and purified by well-known purification methodsincluding ammonium sulfate precipitation, acid extraction, anion orcationic exchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a secretedprotein or peptide that can be further purified to produce desiredamounts of secreted protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe secreted protein or secreted protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native secreted protein is useful forassaying compounds that stimulate or inhibit secreted protein function.

Host cells are also useful for identifying secreted protein mutants inwhich these functions are affected. If the mutants naturally occur andgive rise to a pathology, host cells containing the mutations are usefulto assay compounds that have a desired effect on the mutant secretedprotein (for example, stimulating or inhibiting function) which may notbe indicated by their effect on the native secreted protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a secreted proteinand identifying and evaluating modulators of secreted protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the secreted proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the secreted protein to particularcells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, secreted protein activation, and signal transduction, may notbe evident from in vitro cell-free or cell-based assays. Accordingly, itis useful to provide non-human transgenic animals to assay in vivosecreted protein function, including substrate interaction, the effectof specific mutant secreted proteins on secreted protein function andsubstrate interaction, and the effect of chimeric secreted proteins. Itis also possible to assess the effect of null mutations, that is,mutations that substantially or completely eliminate one or moresecreted protein functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 4 <210> SEQ ID NO 1 <211> LENGTH: 3186<212> TYPE: DNA <213> ORGANISM: Human <400> SEQUENCE: 1ctctgcagct cagcatggct agggtactgg gagcacccgt tgcactgggg tt#gtggagcc     60tatgctggtc tctggccatt gccacccctc ttcctccgac tagtgcccat gg#gaatgttg    120ctgaaggcga gaccaagcca gacccagacg tgactgaacg ctgctcagat gg#ctggagct    180ttgatgctac caccctggat gacaatggaa ccatgctgtt ttttaaaggg ga#gtttgtgt    240ggaagagtca caaatgggac cgggagttaa tctcagagag atggaagaat tt#ccccagcc    300ctgtggatgc tgcattccgt caaggtcaca acagtgtctt tctgatcaag gg#ggacaaag    360tctgggtata ccctcctgaa aagaaggaga aaggataccc aaagttgctc ca#agatgaat    420ttcctggaat cccatcccca ctggatgcag ctgtggaatg tcaccgtgga ga#atgtcaag    480ctgaaggcgt cctcttcttc caaggccatg gacacaggaa tgggactggc ca#tgggaaca    540gtacccacca tggccctgag tatatgcgct gtagcccaca tctagtcttg tc#tgcactga    600cgtctgacaa ccatggtgcc acctatgcct tcagtgggac ccactactgg cg#tctggaca    660ccagccggga tggctggcat agctggccca ttgctcatca gtggccccag gg#tccttcag    720cagtggatgc tgccttttcc tgggaagaaa aactctatct ggtccagggc ac#ccaggtat    780atgtcttcct gacaaaggga ggctataccc tagtaagcgg ttatccgaag cg#gctggaga    840aggaagtcgg gacccctcat gggattatcc tggactctgt ggatgcggcc tt#tatctgcc    900ctgggtcttc tcggctccat atcatggcag gacggcggct gtggtggctg ga#cctgaagt    960caggagccca agccacgtgg acagagcttc cttggcccca tgagaaggta ga#cggagcct   1020tgtgtatgga aaagtccctt ggccctaact catgttccgc caatggtccc gg#cttgtacc   1080tcatccatgg tcccaatttg tactgctaca gtgatgtgga gaaactgaat gc#agccaagg   1140cccttccgca accccagaat gtgaccagtc tcctgggctg cactcactga gg#ggccttct   1200gacatgagtc tggcctggcc ccacctccta gttcctcata ataaagacag at#tgcttctt   1260cgcttctcac tgaggggcct tctgacatga gtctggcctg gccccacctc cc#cagtttct   1320cataataaag acagattgct tcttcacttg aatcaaggga ccttggtcgt ga#aacaatct   1380tctttctttg agttgaaaag ttagcacttc tcctttgagg gtgtcgagct ca#aacaaggc   1440tgtgagaaac aagggagggg agcactaagg ggcaaaccta tctctgcgca ga#tgattctt   1500aggtccagat cataaactag ctctttgcag actatctaca catagtgggg gg#aaagagaa   1560ccagagtcgg aagaggaaca gctgagttta tacagcaagt aagaggtgga gc#taggactc   1620tgattcaact tgctggtaga tggccacaac ccagccgcaa ggcatcagaa ac#aacagggc   1680ctggggcaac tatgcatgtg caaagaggat tggctcagag ttgtggggta gg#aggtccaa   1740tctgggggac ctcaaattat ggttctgggt gattcaagta acaccactca tg#gcttgtgt   1800tgccatgagt taggcatgac aagtggaatg aagttgaagt ggggaaacag aa#atacacca   1860gctgtgtgtc agaggcaagc tggagagaga gaagaaagaa tgaatggcac ca#tggagcac   1920atttgcagaa cacagtccct gggagtcttg ctggagcctc aggagctttg ct#ggcacaga   1980ggatctggcc tacccaatta gcctcctggg tatctgcacc atctagacca gc#aaatgtca   2040ctggcaagga ggttgcagtg cttggttatt ttctggtcat aaactggtga ag#gctttggg   2100ttccaaattt gctgacagct gtttaactgg gaattgggcc tagactatag gt#agctatgt   2160ctcagacaag gccctattcc tccactgcct ttacaaccca gctgaggttg ga#ggctggct   2220tgtttcagcc tcaaaaaata gcctgagttt ccagcagagg gcccttattc tg#agcttccg   2280tgtcctagcc tcattttcct ttcctgtaaa atagacacaa tgccacccac ct#tccagtga   2340caatgaatat agactcaaac ccatcccttg aactgtcttg ggaaggggct ct#ggacgtag   2400acccagactg tggctcatgg cctcatgtga tctggagtca gcccctccca ac#ctgtcagc   2460catttgctcc gtaggacttt gatgggtaga gtagtagcta acaagctctg ac#tgtcacac   2520aaggctttgt actgggaggc caggctatag agtggctcca gcttaaaggg ct#gggagctg   2580ggggacagtg tctcagatta gggtctaact aggaagttga ctggagctga ga#acagaggt   2640taggggccaa gcagcagggt tgtgggtcta ctccttagga gcaccttgag ct#ttactttt   2700cattcctaat ggtgtcttgg atggctaccc tcacggggtt ggctgctagt ct#aaggggtg   2760gagacaagga cagagtttca ggtctggtcc ttatcaagtt catgcactac ac#ttgggacc   2820actgctgcat catgccaggg agcctagagg tgtctaaaca gttatccaac aa#ctgtgata   2880cccaaggtta actttctctt gttttcagag gcagggagta ctaagtctcc cc#tttctcct   2940ttcctcccac gtgttctctt gcagggaatc ctctagcttg tctccaggga ac#tcccagaa   3000atggtttgtt tcagtcagtt taggctgcta taagagaata tcttagagtg gg#taatctat   3060cagcaatagg aatttattgt tcacaattct ggaggctgga aaatccaaga tc#aaggctcc   3120agcaggttca gtgtctgctg agtgcttgtt ctgcttcgaa gatggcacct tt#ttgctgtg   3180 ttctca                  #                  #                   #         3186 <210> SEQ ID NO 2 <211> LENGTH: 391<212> TYPE: PRT <213> ORGANISM: Human <400> SEQUENCE: 2Met Ala Arg Val Leu Gly Ala Pro Val Ala Le #u Gly Leu Trp Ser Leu 1               5   #                10   #                15Cys Trp Ser Leu Ala Ile Ala Thr Pro Leu Pr #o Pro Thr Ser Ala His            20       #            25       #            30Gly Asn Val Ala Glu Gly Glu Thr Lys Pro As #p Pro Asp Val Thr Glu        35           #        40           #        45Arg Cys Ser Asp Gly Trp Ser Phe Asp Ala Th #r Thr Leu Asp Asp Asn    50               #    55               #    60Gly Thr Met Leu Phe Phe Lys Gly Glu Phe Va #l Trp Lys Ser His Lys65                   #70                   #75                   #80Trp Asp Arg Glu Leu Ile Ser Glu Arg Trp Ly #s Asn Phe Pro Ser Pro                85   #                90   #                95Val Asp Ala Ala Phe Arg Gln Gly His Asn Se #r Val Phe Leu Ile Lys            100       #           105       #           110Gly Asp Lys Val Trp Val Tyr Pro Pro Glu Ly #s Lys Glu Lys Gly Tyr        115           #       120           #       125Pro Lys Leu Leu Gln Asp Glu Phe Pro Gly Il #e Pro Ser Pro Leu Asp    130               #   135               #   140Ala Ala Val Glu Cys His Arg Gly Glu Cys Gl #n Ala Glu Gly Val Leu145                 1 #50                 1 #55                 1 #60Phe Phe Gln Gly His Gly His Arg Asn Gly Th #r Gly His Gly Asn Ser                165   #               170   #               175Thr His His Gly Pro Glu Tyr Met Arg Cys Se #r Pro His Leu Val Leu            180       #           185       #           190Ser Ala Leu Thr Ser Asp Asn His Gly Ala Th #r Tyr Ala Phe Ser Gly        195           #       200           #       205Thr His Tyr Trp Arg Leu Asp Thr Ser Arg As #p Gly Trp His Ser Trp    210               #   215               #   220Pro Ile Ala His Gln Trp Pro Gln Gly Pro Se #r Ala Val Asp Ala Ala225                 2 #30                 2 #35                 2 #40Phe Ser Trp Glu Glu Lys Leu Tyr Leu Val Gl #n Gly Thr Gln Val Tyr                245   #               250   #               255Val Phe Leu Thr Lys Gly Gly Tyr Thr Leu Va #l Ser Gly Tyr Pro Lys            260       #           265       #           270Arg Leu Glu Lys Glu Val Gly Thr Pro His Gl #y Ile Ile Leu Asp Ser        275           #       280           #       285Val Asp Ala Ala Phe Ile Cys Pro Gly Ser Se #r Arg Leu His Ile Met    290               #   295               #   300Ala Gly Arg Arg Leu Trp Trp Leu Asp Leu Ly #s Ser Gly Ala Gln Ala305                 3 #10                 3 #15                 3 #20Thr Trp Thr Glu Leu Pro Trp Pro His Glu Ly #s Val Asp Gly Ala Leu                325   #               330   #               335Cys Met Glu Lys Ser Leu Gly Pro Asn Ser Cy #s Ser Ala Asn Gly Pro            340       #           345       #           350Gly Leu Tyr Leu Ile His Gly Pro Asn Leu Ty #r Cys Tyr Ser Asp Val        355           #       360           #       365Glu Lys Leu Asn Ala Ala Lys Ala Leu Pro Gl #n Pro Gln Asn Val Thr    370               #   375               #   380Ser Leu Leu Gly Cys Thr His 385                 3 #90 <210> SEQ ID NO 3<211> LENGTH: 13737 <212> TYPE: DNA <213> ORGANISM: Human<400> SEQUENCE: 3tccctctccc caggcaggcc cagcaaaatc tgtaggattc agacagggtt ct#gacagctg     60aagacaagtt gttgaggaaa ttcctgatgg aggatcatgg ggtgctcagg ag#ggagaata    120taaggtttca gaggctgaga gggaaagaaa aggtgagggg gagtcttaga at#agtggctc    180ccattgccca acacccagaa agaagacatg ccctgcaatg gggagaaggt ga#gtatgaga    240cattggctgt agcagcgatg gcattgccca ggctgccaag gactcagaga gt#ccagcctt    300gcccactgac ctatgaggag ggaatgatgt tcacagcaca ttttcattcg ta#agtcagga    360gaggacattg agcctgatgg cagaggcctg gtgacatgtt gttccagagg tt#ccggaatg    420tgtgttttcc tgttggaagg aaacttcgca gagtagaaaa gggatctgag ac#ttttggta    480agattatata tgggactgtc aggggtctgg agccatctgt gagggatcag gg#ccctttca    540gccttggcta gggagcaggg gtcctggaac ttcatcctgg cccatagctg ag#tctgccca    600taattctttt ctgactcact aggcaaatct cacacagaaa tggggcagct tt#gggagtgg    660gcccaggaag tactgaggat agcaggtgag atcccaggaa gagatggatg tg#gggccgag    720acactggaga gagaaacagg actgtcagat aaagggcgtc tgtgactcct ag#atctcatt    780atgcctacta ccataaccta cccccaattc ctaatattct cctaccctag ag#ggggggaa    840attgtcagaa atttggctgc aacactagca acactactca gtacttgaaa tg#catttttg    900catttttttc attcaacaaa tatttctgga acaactctta tatgccaggc ac#tattttag    960gagtcaggga tatataatgg taaacaagac aggcaaaaca aagcaaagca ac#aacaacca   1020tcaccagata agtagacaga tgaaagaatt tcaagtttta gtaagtaaaa ta#aaacaagc   1080aagggtctga aatggctaga taaggcggtc aagaaaggct tcattgagaa gg#tagcattt   1140aagcaggagt cagctagaaa tattgtgaaa ttccagttac agttctattt gt#tctgggtt   1200ggttaaataa agctttttcc cccaaggtgg aaactaccaa gaaagactaa tt#actagtag   1260tggtggtgct ctctggaaga gagacacctc ctgtttctgc ctcattactg tc#aacccttc   1320acttccaggc actttttgca aagccctttg ccagtcaggg aaggcgagag gc#tgggcatg   1380gggcttggac atttgacaac agtgagacat tattgtcccc agactcacta gc#ccaagggt   1440aaagctgaag aggcttgggc atgccccaga aaggcccctg atgaagcttg ga#aaaagctg   1500ttctctgagt atttctaagt aagtttatct gtgtgtgtgg ttactaaaag ta#gtaagtat   1560tgctgtctct agctgcctta gagcagggct tgacacagta cacagcaata tt#agttccct   1620ccttttctca cctcccccat tgtggagata aactcaatca caaaaggtga tc#ctcagtct   1680actcacttcc ctgacttatg gatgcctgga cccattgcca gtgtgagagt ca#cagctgga   1740cgtcagcagt gtagcccagt tactgcttga aaattgctga agggggttgg gg#ggcagctg   1800ccgggaaaaa ggagtcttgg attcagattt ctgtccagac cctgacctta tt#tgcagtga   1860tgtaatcagc caatattggc ttagtcctgg gagacagcac attcccagta ga#gttggagg   1920tgggggtggt gctgctgcca actctatata gggagttcaa ctggtcaccc ag#agctgtcc   1980tgtggcctct gcagctcagc atggctaggg tactgggagc acccgttgca ct#ggggttgt   2040ggagcctatg ctggtctctg gccattgcca cccctcttcc tccgtgagta aa#gctgggac   2100tagaagcgaa ggattgagtt ctgggctagg gtaaggtagg gccagttttt ag#gcctcggt   2160caaatttggg gtcaggggct atgggaaagg gatcggtccc aatggatcaa ga#tatctatt   2220ttgttctccc taggactagt gcccatggga atgttgctga aggcgagacc aa#gccagacc   2280cagacgtgac tggtgaggcc ctgactccct aagtctgtct tatctgtctg gt#tgtgtctc   2340tgcattttat caccttctgg tttttttttt tttttttttt ttttactttg cc#atctccct   2400acctccaccc cagaacgctg ctcagatggc tggagctttg atgctaccac cc#tggatgac   2460aatggaacca tgctgttttt taaaggtagg agggactgag gttagggcgt tt#aggacctt   2520agacttactc tccttcacaa agggtgtccc tgtctgtggg aggtcttagg aa#ttatctga   2580tggtatcact gacagcttct ctcaagctat ctcagtaggt caaaggtttc tc#actgggcc   2640cctcagtgag tgtgggtttt ttcaggggag tttgtgtgga agagtcacaa at#gggaccgg   2700gagttaatct cagagagatg gaagaatttc cccagccctg tggatgctgc at#tccgtcaa   2760ggtcacaaca gtgtctttct gatcaaggta ctgctgggcc aaaatcaggg cc#aggctgga   2820aagggctgga atcgacactg gggacccttc ccccaaatgg ccttggcatg ga#gcccatag   2880caataggtag cagatttctt tcccatgtgc cctcctttcc tgtaaaagct tg#ggctaagg   2940gagtgtgcat gcgtgtgggc ctggcaggtg caccatccag tggctgttct tc#agtcctag   3000tcttagttct acaccgctct gctgtacctc acactgctgg ccatcctttt tt#tctctggc   3060aattgcttcc cttgccttcc atgaccctgt atcaagtcct cttcataggg ca#aggcaagt   3120tgttcccaac acaatggcac ctggctagaa gagcatgtgg agcatgaaat cc#agtctgct   3180gtgctcacca agtcccatgt gacccaggct gtgtctgctc agaggaaggg gt#gccttttc   3240ctaccttgcc aaaggtgctg tgtggttggg gaagtcctga ctgtcggctt tg#ttttccct   3300cctgcctctt ttctctctct tctcaaatgt ctcattctat ctcaaccagt tc#cctaatgt   3360tccttgggga tccatcctag cctttccata taccttccct cagtgatctc aa#ccatcacc   3420ttggctctga ggaatatcta tgctgtggac actggatcta gatctacttt ct#gagctcca   3480gacatctctt tccaattgta tgttctacag gcacctaaaa ttcagcatcc cc#caaactaa   3540gctttgcatc ttctttacaa accaaccttt cctcctgtgt ttcctgtttc ag#taaatgac   3600cccaaaatgt gcctgattac tacaaaccaa gtgcacacag ggtctcatga tc#tgggcctt   3660ggttatcttc tcaggtttat ctcctcccct gccacattca ctgtgtgcca gc#catacgaa   3720tctacatgag gttggagcac actgcttcct catgtttggg ctctgcatgc tg#ctccctct   3780gctggtaaca ccctttcctc acttgtcaac ctggaaaatt cctgctgatt tt#tcagctct   3840tgggcccaat gcttcctctt tggtgtgaaa ccttccacaa cttctctagg ca#gacttagg   3900cactctgtct atattctcag tgcactcttt acactacacc ttggtagttg ca#tggctagg   3960attgcaggag tcctttctgc ttttgtacag tgaacttcct gaagtgaaag ac#agagtctt   4020gttatcctca gtgcctctca caatgcctgg catatagtag ttattcagtg ac#tgtttctt   4080ggatgaatga atgaatgaat aaataaatga agaaatgaat gaagaaataa cg#tatgggtg   4140attgcaggat gaacagttgt ggatatgttt gtcaacactg atagtgttgc ag#ataaatgt   4200gccacaggag tgtctgggta cagagctaga ggcatgtgtg ttatagtaat ag#tgactgga   4260tttgcacaaa ctgagagtgt gtaatgtgca aaaggacagc acattgttgt cc#acagatgg   4320actgagaatg tgtagggcca cagaaggata tcgtataagc acagtagata aa#aaatgtgt   4380gtaaatgcag agtggcagta tctggggatg cacagtcaaa aagagagtac tt#ttgaatgc   4440agggggacaa agtctgggta taccctcctg aaaagaagga gaaaggatac cc#aaagttgc   4500tccaagatga atttcctgga atcccatccc cactggatgc agctgtggaa tg#tcaccgtg   4560gagaatgtca agctgaaggc gtcctcttct tccaaggtca gtccaggctg ga#atccaaga   4620acctggagta gtggtgggtt ggtagtgatg ccagtagtga tggtgatagt gg#tagtgatg   4680gtggtggtgg agccactatg tggcttttta aggaagggaa atagagaagc ca#cgtatggt   4740ctagaggtca cgtgagggaa ggagaggaag tcattctggt gaaggcaact gt#gtgtaatt   4800ctgtgtgaat agtccctcat tgttccccat gacccttagg acaaatctac cc#tctttagt   4860cttacataca agtctctcca tggccaaatc cctattggcc cttcagcttt ga#cttttatt   4920atacttttac cttaacacta agctccagaa accctatgct attctctgta ca#ctcagttt   4980gctccatgct ttggaatctt tcctctctct ggggttccat ctctccttgt gt#gcctttta   5040attcctactt cagatttcac tttaagtatc atcttccctg ggaagttttc cc#agactctc   5100cccactgcct ttgctgagct gatcctgtgt gttttgctgc tgaattttgg tg#tatgatca   5160ccctccttta gccatctctc tgatggctgt gagctccatg tggtcagtac ca#ttatctgg   5220cccatcctgg gacccagaga aagcacaaag gagggcgtaa cccggtctca cc#aaatgcct   5280gttgattgat tggacaaagg tgaccgcgag tggttctggg acttggctac gg#gaaccatg   5340aaggagcgtt cctggccagc tgttgggaac tgctcctctg ccctgagatg gc#tgggccgc   5400tactactgct tccagggtaa ccaattcctg cgcttcgacc ctgtcagggg ag#aggtgcct   5460cccaggtacc cgcgggatgt ccgagactac ttcatgccct gccctggcag ag#gtgagaaa   5520gccctagcac ttgagacctg tcagaattca tccactttcc ctgagcttgt gg#atctcacg   5580tgtcctagct ctcactttaa ctccgtgttg cgacaccttg gcccttaatc ta#gccccatt   5640tccattctgg attttcccat tgccctcata tggggaaacc cacaccccac ta#accccagc   5700catctcttcc accttggacc tcactctgac ctctggcctc cttctgtgtt ct#cctcaccc   5760atttctctct ccaggccatg gacacaggaa tgggactggc catgggaaca gt#acccacca   5820tggccctgag tatatgcgct gtagcccaca tctagtcttg tctgcactga cg#tctgacaa   5880ccatggtgcc acctatgcct tcagtggtga gagatgcccc caactccccc aa#tgtgctct   5940cacatctctt ttacttgtat ctcccatcct tgacacattt ctccattgtc at#cactgtgt   6000cacttatttt gtcccctctg tccccatcct tctgcatgcc cttctgcatc cc#tcatctct   6060gaggcatatt tctcaatctt gtctgtcacg gcccaagccc ctaacttcat ct#acctgtct   6120accatctact cccatggctg tgccccctgt ggacctctct gggcccctat ga#ctccttgt   6180gttctccttg ctcaatgccc tgctgagccc tctggctctc ccttgctccc tg#gacctcta   6240tgtgtctctg tacctccttg cctccctttg ttcttgcata tctttctgag tc#ctctggct   6300ccccctgatt tatcctcaga actccatctt gtttcaggtt cctggttcct at#gtccagac   6360ccctgggcat agcactgcct ggggatgaga tgttctcatt gctgagaacc ag#ctgagaag   6420tgttgggtac tttagacctt tagaggctgg cttcactagc ctctggaggt tt#ctcctctg   6480agtagccaat ggagataccc ctcccttgac ccgtggcatc aattggtaaa ag#ccatctaa   6540taatacctag ggctgttctg agttcagtca ggcagtaaat agtcatgctg ca#cagttgag   6600aatatcccca agaggagtga gcaaccacat cacatccaac ctgagatata tg#tataatta   6660ggacagtggt aagaatataa aatcgtgaaa atattttttt cacacaaaat tt#ttttggct   6720cctgaccctt ggacaaattt gaccagttat gactatcaag ttctgttgaa aa#atacatca   6780ccacatggag agcaaatctc cacagcagga ttgcacacta taataagaac at#acagctaa   6840gatgaaacac acacctgtag tgaaaataca acattaaact gagaacatac gc#catagtaa   6900gaacacataa gtatcaagag aacacacagc catggtggga gcccattggg ag#gacacaca   6960gacaaagtga aatgcagaaa gagagagaga gtgagtgaga gattgtgaaa ac#agggccac   7020aggaaacaca cagaaataga gagagacacc aagccatcta gagatcacag aa#cttcatgg   7080ccatgtggcc ataatgagaa tgctactgaa ctcctaaatg aaaaatgtca tg#tatgttcc   7140atagctgttg agagagccca cagcatggag agaacacctt atattaaaaa ta#cccaggcc   7200gggcgtggtg agtcacgcct gtaatcctag cactttggga ggctgaggca gg#tggattgc   7260ttgagcggct tgagcctagg agtttgagac cagcctgggc aacatggcaa aa#cctcatct   7320ctacaaaaaa tataaaaatt agtcgggtgt ggtagtgcgt tcctatagtc cc#atctactt   7380cagaggctga gcccggaagg tcgaggcttc agtgagccgt gatcgtgcta ct#gcactcca   7440gcctgggtga cagagtgaga ccatgtctca aaaaaaacaa aaacaaaaaa ca#aaacaaaa   7500caaacaaaca aacaaaaaac ccatatatat atatatatac ctagctgagg tg#agaatgca   7560ctattttggt aaaatcacca acatgaccca gctacagcat ggggcagtcc ct#cccctctc   7620actggtaaat ttttctttct ctgactcaca gttttgttgt tgttgttgct gt#tgtttgag   7680atggagtctc actctgtcac ccaggctgga gtgcaatggc gcaatcttgg tt#cactgcaa   7740cctctgcctc ctgggttcaa gcgatcctcc tgcctcagcc tcccgtatag ct#gggactac   7800aggcgcatac caccatgcct ggctaatttt tgtatttttt tttgggttac aa#tgtactat   7860ttattaattt aatttttgta tttttagtag agatagggtt tcaccatgtt gg#ccaggctg   7920gtctcgaact cctgacctca ggtgatccgc ctgcctcggc ctcccaaagt gc#taggatta   7980caggcatgag caaccacgcc tggcccctca taggttttta tctattctct tt#gcttcttc   8040acaactttgg cttgcacgtg gaccatcatg ttctctccac tttctcacta ct#tcatgatc   8100tttcagtctc agttccaact gatacctccc tcagttgctc ttttttccta gt#aagatttc   8160cagagaggga atctgaatgg cccagtccat attttcagac cacaccacat ta#aagtggtt   8220gattgccagc ctatgtattg gctacattaa tgggttggga actcatcatt ta#cttcattg   8280cacaaagcag catagctctg gttctcaaaa tagggcccct gggccaggtg tg#gtggctca   8340tgcctataat cccaacactg tgggaggccg aggggggcag atcacttgag tc#caggagtt   8400ctagaccagc ctgggcaaca tggtgaaatc tcatctctac taaaaataca aa#aaattagc   8460caggtgtggt ggcatgcacc agtagtccca gctgttcagg aggctgaggt gg#gaggattg   8520ctcgagtgtg ggaggcagag attgcagtga accgtgactg tgcctctgca at#ccagcctg   8580ggtgacagat tgagaccctg tctcaaaaaa caaataaata aaataaaata aa#tatggttc   8640ctgagcaggg taatttcagt gggaaacctc ccaggggagg tggatatgtc ag#tcaccgct   8700gtatactcag tacacggcta ataagagaac ttgtggtagc agcaagaaca ct#aggtattt   8760actcaacaaa tatttgttga gcatctgata agaagtgggc attgtcctag gc#actgagat   8820acagtagtca acatggcaga caagatgcct gccctgacag gctctgctaa ag#tgagagag   8880gacaataaga aagagaaagg aagaaagaga ataattttag gtaatattaa gg#gttgtaaa   8940gaaaataaga caggatagtg ggatagaggt gaggagaatg agggctgtct tc#tgaagaaa   9000tgatttttga gctgagactt cagtgatgag aaggaattaa ccacacgatg tg#ctggagga   9060aaagcatttt agggagggtg agcagcacat acttcaagga atcaagaagg aa#gcctggtg   9120aggctggaac acagagaaag agcaggtggg tgacttgaaa gggcagggac gg#cagtggcc   9180aggttaccta gacctggtaa gggttttcaa ccataaaagg gagtcatcag aa#agtcttga   9240gcagggctgt gatatattct aactcatttt ttataaaaga tcactctgac tt#tttgcaga   9300acataagtta taaaagtaca agcatgtaag caaggaatcc agctagcaat cc#gtgcagtt   9360gtccaaatta gaggtgatga ccgcttggac taggatgata gcagcagagg tg#gtgaggaa   9420tcaccatgat atattttgga ggtagagctg acagcattaa ctaatagcta ag#ataggccg   9480ggtgtggtgg cttacgcctg taatcctagc actttgggag gccaaggcga gt#ggatcacc   9540tgaggtcagg agttcgagac cagcttgacc aacatggtga aacctcgtct ct#actaaaaa   9600tacaaaatta gctgggaatg gtggcacatg cctgtaatct cagcctactt gg#gaggctga   9660ggcaggagaa tcgcttgaac ctgggaggtg aatgttgcag tgagccgaga tt#gcaccatt   9720gcactccagc ctggggaaca agagtgaaac tccgtctcta aataaatgaa tg#aatgaatg   9780atatcagtca gagtagggaa gggaaaagag gcttcaagaa tgactcagct tt#cgtggact   9840cagcaactga gtggctggtg gttttgtttt ctaaaattgg gaaagactag gg#agtgtgtg   9900tgttggtggg gggcagaaat cagtttgggc atattaggtt ttgggtgcct at#tggcaccc   9960cataagcatg tcaggtaggc agctgatttg gagcctaaac ctcaaaggag ag#gtcagtca  10020gagctgacga gaacagattg gaagtcatca gcatatagat ggcatttaaa gc#ccctggac  10080taggtgagat taccaaggaa gtgaaggtag agagagaaga gaagaggccc aa#agtagggg  10140attccaatat ttagatatca ggttgaagaa aagagtagtc aaaaaagata ag#aggaatac  10200tgggagagtc aggtgtcaca gaagccaagt tccaaaaaaa gacatttaaa gg#agaaggaa  10260gtagtgagca gtccagtgct cctgagaggt agggtcagat gagaacagag aa#ttgaccat  10320gagatttcgc aaattggaga atactagcaa cctggataag aacaatttca at#ggttgagg  10380gaaacagaag tgtaattgaa gaggattgag gaaaaaagac aaatgggagc ct#agataatt  10440ccttaataag ttgttgtgaa aagaggagaa gaaaaacggg gtgctagccc ag#ctactccc  10500tcactcttcc accacctcat agggagagac tggagaacac agccagagtg ag#aacattca  10560gtagaagtgg tgcttccttt ttaagttctg gacactgtat ttcattatct at#aaccgcat  10620ctctgtacat ggacacctga aatccttagg gagtgcccgc caaccccatg at#gttggcct  10680tacctggaaa cttagccact gttttccaca cttgcctttc tttcaggcac ct#gctgattc  10740cagtttcagc cagggcacag tgcccaacat tgctgaccaa gtcttgctct at#ttctcctt  10800ctcacctggc ctcttccatc ttggcctctg gatgcattct ctccctctca tg#actcattt  10860ctgcattcat cactagcctc ttctctgcct gggcttctgc cagcggccct ag#agcaacct  10920atggtattcc acagggaccc actactggcg tctggacacc agccgggatg gc#tggcatag  10980ctggcccatt gctcatcagt ggccccaggg tccttcagca gtggatgctg cc#ttttcctg  11040ggaagaaaaa ctctatctgg tccaggtgtg tattggggga gaggcttgag gt#agagactg  11100ggacaagcat atccaactct gtatttatta ccatcctttg tcctccaggg ca#cccaggta  11160tatgtcttcc tgacaaaggg aggctatacc ctagtaagcg gttatccgaa gc#ggctggag  11220aaggaagtcg ggacccctca tgggattatc ctggactctg tggatgcggc ct#ttatctgc  11280cctgggtctt ctcggctcca tatcatggca ggtgaggggc ttctgggtgc tt#agagggca  11340gcttgttctg ctacctgtct gtggcataga tccccaccag ggcatgagaa gg#cctaggtc  11400aggatcccca gggcatgaga aggcctaggt caggatcccc atgacatgga ag#ccatgcta  11460tgtttggtgc cttctcccca ggacggcggc tgtggtggct ggacctgaag tc#aggagccc  11520aagccacgtg gacagagctt ccttggcccc atgagaaggt agacggagcc tt#gtgtatgg  11580aaaagtccct tggccctaac tcatgttccg ccaatggtcc cggcttgtac ct#catccatg  11640gtcccaattt gtactgctac agtgatgtgg agaaactgaa tgcagccaag gc#ccttccgc  11700aaccccagaa tgtgaccagt ctcctgggct gcactcactg aggggccttc tg#acatgagt  11760ctggcctggc cccacctcct agttcctcat aataaagaca gattgcttct tc#gcttctca  11820ctgaggggcc ttctgacatg agtctggcct ggccccacct ccccagtttc tc#ataataaa  11880gacagattgc ttcttcactt gaatcaaggg accttggtcg tgaaacaatc tt#ctttcttt  11940gagttgaaaa gttagcactt ctcctttgag ggtgtcgagc tcaaacaagg ct#gtgagaaa  12000caagggaggg gagcactaag gggcaaacct atctctgcgc agatgattct ta#ggtccaga  12060tcataaacta gctctttgca gactatctac acatagtggg gggaaagaga ac#cagagtcg  12120gaagaggaac agctgagttt atacagcaag taagaggtgg agctaggact ct#gattcaac  12180ttgctggtag atggccacaa cccagccgca aggcatcaga aacaacaggg cc#tggggcaa  12240ctatgcatgt gcaaagagga ttggctcaga gttgtggggt aggaggtcca at#ctggggga  12300cctcaaatta tggttctggg tgattcaagt aacaccactc atggcttgtg tt#gccatgag  12360ttaggcatga caagtggaat gaagttgaag tggggaaaca gaaatacacc ag#ctgtgtgt  12420cagaggcaag ctggagagag agaagaaaga atgaatggca ccatggagca ca#tttgcaga  12480acacagtccc tgggagtctt gctggagcct caggagcttt gctggcacag ag#gatctggc  12540ctacccaatt agcctcctgg gtatctgcac catctagacc agcaaatgtc ac#tggcaagg  12600aggttgcagt gcttggttat tttctggtca taaactggtg aaggctttgg gt#tccaaatt  12660tgctgacagc tgtttaactg ggaattgggc ctagactata ggtagctatg tc#tcagacaa  12720ggccctattc ctccactgcc tttacaaccc agctgaggtt ggaggctggc tt#gtttcagc  12780ctcaaaaaat agcctgagtt tccagcagag ggcccttatt ctgagcttcc gt#gtcctagc  12840ctcattttcc tttcctgtaa aatagacaca atgccaccca ccttccagtg ac#aatgaata  12900tagactcaaa cccatccctt gaactgtctt gggaaggggc tctggacgta ga#cccagact  12960gtggctcatg gcctcatgtg atctggagtc agcccctccc aacctgtcag cc#atttgctc  13020cgtaggactt tgatgggtag agtagtagct aacaagctct gactgtcaca ca#aggctttg  13080tactgggagg ccaggctata gagtggctcc agcttaaagg gctgggagct gg#gggacagt  13140gtctcagatt agggtctaac taggaagttg actggagctg agaacagagg tt#aggggcca  13200agcagcaggg ttgtgggtct actccttagg agcaccttga gctttacttt tc#attcctaa  13260tggtgtcttg gatggctacc ctcacggggt tggctgctag tctaaggggt gg#agacaagg  13320acagagtttc aggtctggtc cttatcaagt tcatgcacta cacttgggac ca#ctgctgca  13380tcatgccagg gagcctagag gtgtctaaac agttatccaa caactgtgat ac#ccaaggtt  13440aactttctct tgttttcaga ggcagggagt actaagtctc ccctttctcc tt#tcctccca  13500cgtgttctct tgcagggaat cctctagctt gtctccaggg aactcccaga aa#tggtttgt  13560ttcagtcagt ttaggctgct ataagagaat atcttagagt gggtaatcta tc#agcaatag  13620gaatttattg ttcacaattc tggaggctgg aaaatccaag atcaaggctc ca#gcaggttc  13680agtgtctgct gagtgcttgt tctgcttcga agatggcacc tttttgctgt gt#tctca     13737 <210> SEQ ID NO 4 <211> LENGTH: 462 <212> TYPE: PRT<213> ORGANISM: Human <400> SEQUENCE: 4Met Ala Arg Val Leu Gly Ala Pro Val Ala Le #u Gly Leu Trp Ser Leu 1               5   #                10   #                15Cys Trp Ser Leu Ala Ile Ala Thr Pro Leu Pr #o Pro Thr Ser Ala His            20       #            25       #            30Gly Asn Val Ala Glu Gly Glu Thr Lys Pro As #p Pro Asp Val Thr Glu        35           #        40           #        45Arg Cys Ser Asp Gly Trp Ser Phe Asp Ala Th #r Thr Leu Asp Asp Asn    50               #    55               #    60Gly Thr Met Leu Phe Phe Lys Gly Glu Phe Va #l Trp Lys Ser His Lys65                   #70                   #75                   #80Trp Asp Arg Glu Leu Ile Ser Glu Arg Trp Ly #s Asn Phe Pro Ser Pro                85   #                90   #                95Val Asp Ala Ala Phe Arg Gln Gly His Asn Se #r Val Phe Leu Ile Lys            100       #           105       #           110Gly Asp Lys Val Trp Val Tyr Pro Pro Glu Ly #s Lys Glu Lys Gly Tyr        115           #       120           #       125Pro Lys Leu Leu Gln Asp Glu Phe Pro Gly Il #e Pro Ser Pro Leu Asp    130               #   135               #   140Ala Ala Val Glu Cys His Arg Gly Glu Cys Gl #n Ala Glu Gly Val Leu145                 1 #50                 1 #55                 1 #60Phe Phe Gln Gly Asp Arg Glu Trp Phe Trp As #p Leu Ala Thr Gly Thr                165   #               170   #               175Met Lys Glu Arg Ser Trp Pro Ala Val Gly As #n Cys Ser Ser Ala Leu            180       #           185       #           190Arg Trp Leu Gly Arg Tyr Tyr Cys Phe Gln Gl #y Asn Gln Phe Leu Arg        195           #       200           #       205Phe Asp Pro Val Arg Gly Glu Val Pro Pro Ar #g Tyr Pro Arg Asp Val    210               #   215               #   220Arg Asp Tyr Phe Met Pro Cys Pro Gly Arg Gl #y His Gly His Arg Asn225                 2 #30                 2 #35                 2 #40Gly Thr Gly His Gly Asn Ser Thr His His Gl #y Pro Glu Tyr Met Arg                245   #               250   #               255Cys Ser Pro His Leu Val Leu Ser Ala Leu Th #r Ser Asp Asn His Gly            260       #           265       #           270Ala Thr Tyr Ala Phe Ser Gly Thr His Tyr Tr #p Arg Leu Asp Thr Ser        275           #       280           #       285Arg Asp Gly Trp His Ser Trp Pro Ile Ala Hi #s Gln Trp Pro Gln Gly    290               #   295               #   300Pro Ser Ala Val Asp Ala Ala Phe Ser Trp Gl #u Glu Lys Leu Tyr Leu305                 3 #10                 3 #15                 3 #20Val Gln Gly Thr Gln Val Tyr Val Phe Leu Th #r Lys Gly Gly Tyr Thr                325   #               330   #               335Leu Val Ser Gly Tyr Pro Lys Arg Leu Glu Ly #s Glu Val Gly Thr Pro            340       #           345       #           350His Gly Ile Ile Leu Asp Ser Val Asp Ala Al #a Phe Ile Cys Pro Gly        355           #       360           #       365Ser Ser Arg Leu His Ile Met Ala Gly Arg Ar #g Leu Trp Trp Leu Asp    370               #   375               #   380Leu Lys Ser Gly Ala Gln Ala Thr Trp Thr Gl #u Leu Pro Trp Pro His385                 3 #90                 3 #95                 4 #00Glu Lys Val Asp Gly Ala Leu Cys Met Glu Ly #s Ser Leu Gly Pro Asn                405   #               410   #               415Ser Cys Ser Ala Asn Gly Pro Gly Leu Tyr Le #u Ile His Gly Pro Asn            420       #           425       #           430Leu Tyr Cys Tyr Ser Asp Val Glu Lys Leu As #n Ala Ala Lys Ala Leu        435           #       440           #       445Pro Gln Pro Gln Asn Val Thr Ser Leu Leu Gl #y Cys Thr His    450               #   455               #   460

That which is claimed is:
 1. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a neucleotide sequence that encodes a polyeptide having an aminoacid sequence comprising SEQ ID NO:2; (b) a nucleotide sequenceconsisting of SEQ ID) NO:l; (c) a nucleotide sequence consisting of SEQID NO:3; and (d) a nucleotido sequence that is completely complementaryto a nucleotide sequence of(a)-(c).
 2. An isolated polynucleotide,wherein the nucleotide sequence of said polynucleotide consists of SEQID NO: 1 or the complement thereof.
 3. An isolated polynucleotide,wherein the nucleotide sequence of said polynucleotide consists of SEQID NO:3 or the complement thereof.
 4. A vector comprising the nucleicacid molecule of claim
 1. 5. The vector of claim 4, wherein said vectoris selected from the group consisting of a plasmid, a virus, and abacteriophage.
 6. The vector of claim 4, wherein said isolated nucleicacid molecule encodes a polypeptide comprising SEQ ID NO:2 and isinserted into said vector in proper orientation and correct readingframe such that a polypeptide comprising SEQ ID NO:2 is expressed by acell transformed with said vector.
 7. The vector of claim 6, whereinsaid isolated nucleic acid molecule is operatively linked to a promotersequence.
 8. An isolated host cell containing the vector of claim
 4. 9.A process for producing a polypeptide comprising SEQ ID NO:2, theprocess comprising culturing the host cell of claim 8 under conditionssufficient for the production of said polypeptide, and recovering saidpolypeptide.